Halo enol lactones can act as enzyme-activated irreversible inhibitors or suicide substrates for (alpha)-chymotrypsin. Acyl transfer to the active site serine generates a halo methyl ketone that remains tethered in the catalytic site during the lifetime of the acyl enzyme, available for reaction with an enzymatic nucleophile. To investigate the structural determinants for chymotrypsin suicide inactivation with halo enol lactones, a series of lactones were prepared differing in ring size (6-membered valerolactones and 5-membered butyrolactones), and in the nature of the aromatic substituent (phenyl and naphthyl) and the halogen. The kinetic parameters for the inactivation process were determined for each inhibitor and it was found that the valerolactones were more potent inactivators than the butyrolactones; the naphthyl-substituted lactones had both a greater binding affinity and efficiency of inactivation than the phenyl-substituted ones; and the nature of the halogen had relatively little effect. Several of the valerolactones were resolved into their enantiomers and it was found that the nature of the stereoisomer had only a small effect on the inactivation parameters. The possible involvement of a paracatalytic mechanism for inactivation (generation of a free, rather than active site-bound inactivating species) was investigated by comparing the inactivation efficiencies of the lactones with that of the halo methyl keto acid hydrolysis products. The possible involvement of the free keto acid in chymotrypsin inactivation is ruled out by experiments involving selective scavenging. The long-term inactivation of chymotrypsin requires the presence of the halogen substituent and appears to involve an alkylation rather than an acylation reaction (hydrazine resistant). Furthermore, a 1:1 lactone: enzyme stoichiometry of inactivation is demonstrated using a ('14)C-labeled valerolactone. These studies are consistent with the proposed mechanism of inactivation and show that relatively small changes in the halo enol lactone structure result in significant differences in the potency and efficiency of enzyme inactivation, with some of the lactones approaching ideal behavior as suicide substrates.